Regulation of mevalonate kinase activity was studied in human skin fibroblasts grown in tissue culture and in rat liver. Phosphomevalonate kinase, another enzyme in the biosynthetic pathway of cholesterol, was also assayed in rat liver. The activity of both enzymes was reduced by over 50 percent in livers of cholesterol-fed rats. In fibroblasts, there was twice as much mevalonate kinase activity following incubation with medium containing solvent-extracted serum as in cells treated with whole serum. There was no such reduction in activity due to serum lipids in fibroblasts derived from a patient with homozygous familial hypercholesterolemia. The activity of mevalonate kinase was increased following incubation with insulin. It appears that the above two enzymes could play a role in the physiological regulation of sterol synthesis.